Optimization of In - vitro Permeation Pattern of Ketorolac Tromethamine Transdermal Patches.

The present study was undertaken to develop a suitable transdermal matrix patch of ketorolac tromethamine with different proportions of polyvinyl pyrrolidone (PVP) and ethyl cellulose (EC) using a D-optimal mixture design. The prepared transdermal patches were subjected to different physicochemical evaluation. The surfacet opography of the patches was examined by scanning electron microscopy (SEM). The drug-polymer interaction studies were performed using Fourier transform infrared spectroscopic (FTIR) technique. A correlation between in - vitro drug-release and in - vitro skin permeation was established and the criterion of desirability was employed to optimize the formulation. The results of the physicochemical characterization and in - vitro permeation of the prepared patches were promising to formulate transdermal patches with PVP/EC combinations.


Introduction
Ketorolac tromethamine (KT) is a nonin the management of moderate to severe pain associated with orthopaedic, gynaecological or urological surgical procedure, act by inhibiting the synthesis of prostaglandins (1). The drug is reported to be 90% oral bioavailable with a very half-life (4-6 h) calls for frequent administration (2), and many adverse effects, such as upper abdominal pain and gastrointestinal ulceration restrict its long term oral use (3). To avoid invasive drug therapy such as injections and to eliminate frequent dosing regimen for maintaining drug blood levels for an extended period of time and also to reduce side effects associated with dosage forms, an alternate non-oral delivery system has been studied to provide controlled release of drug for an extended period. Transdermal drug delivery system (TDDS) pass effect, reducing frequency of administration, potentially decreasing side effects, improved patient compliance sustaining drug delivery and interruption or termination of treatment when necessary (4). Polyvinyl pyrrolidone (PVP) and ethyl cellulose (EC) combinations have been used to formulate matrix type transdermal patches of ketorolac tromethamine. The present work was aimed to study the compatibility and physical characteristics of prepared transdermal patches with different proportion of PVP and EC. Principles of statistical experimental designing were effectively employed to study the effects of formulation variables on in-vitro percutaneous absorption of the drug from its transdermal systems. AD-optimal mixture design was used to study the effects of the mixture components delivery system.

Design of experiment (DOE)
A D-Optimal mixture design was used in development of dosage form. In a mixture test, which is suitable for pharmaceutical formulations, the independent factors are the components of a mixture and the response depends on the relative proportions of each ingredient (5). It involves changing mixture composition and exploring how such changes will affect the properties of the mixture (6). classic mixture designs. These designs are more robust to constraints and can produce complex designs with many design constraints. Unlike standard and classical design of experiments such as factorials and fractional factorials, D-optimal design matrices are usually not orthogonal and effect estimates may be correlated. These types of designs are always an option regardless of the type of the In combined mixture designs, numeric factors (process factors) are also evaluated along with the mixture components.
In the present investigation, PVP (X 1 ) and EC (X 2 ) were selected as mixture components. In a mixture design, the level of a single mixture component can not be changed independently (7) and the sum of the mixture components has to be equal to 100% (8). The restrictions imposed on the mixture component proportions are as follows: The cumulative amount of KT permeated per cm 2 of abdominal mice skin at 24 h (P 24 ), KT released at 8 h (Q 8 ) were chosen as dependent variables (Table 1). Total weight of polymer was w/w per patch. Design-Expert software (Version. 7.1.3, Stat-Ease Inc., (Minneapolis, USA)) was used for the generation and evaluation of the statistical experimental design Preparation of patches Experimental matrix type transdermal patches with varied mixture composition (Table  1) of ketorolac tromethamine were prepared by casting drug dispersion in chloroform over the PVA backing membrane and subsequent evaporation of solvent in an open glass mould. One side of the both-side open-glass mould was wrapped with aluminium foil over which backing membrane was prepared by pouring 4% solution of PVA and dried at 60 C for 6 h. Di-n-butyl phthalate (18% w/w) was added as a plasticizer. Solvent was evaporated slowly at room temperature and controlled evaporation was assured by covering each glass mould with an inverted funnel. After complete removal of the solvent, patches were removed and kept in desiccators till further use.

Physicochemical evaluation of patches Drug-excipient interaction study
The active pharmaceutical ingredient, ketorolac tromethamine and the mixture of drug with excipients were separately mixed with IR grade KBr in the ratio 100 : 1. Then the samples were converted into pellets by applying pressure in a hydraulic press and the pellets were scanned over a wave number range of 4000 to 600 cm -1 in a Magna IR 750 series II (Nicolet, USA) FTIR instrument.

Moisture content
Moisture content of different formulations was determined (Table 2) following the method were weighed and kept in a desiccator containing than 24 h until two successive weights found constant.

Moisture uptake
The patches of different formulations were dried by keeping over activated silica in a desiccator and then taken out of desiccator and exposed to 84% relative humidity produced by taking saturated solution of potassium chloride in a desi ere taken e periodically till two successive weights found constant (Table 2).

Thickness
Thickness of the prepared patches was determined using calibrated eye piece micrometer in a compound microscope. The patches were sliced into pieces; the pieces were then placed vertically on the slide and the thickness of different cross section was measured and average data presented in Table 2. of 2.49 cm of each formulation were weighed individually. The variation of individual weight from the average weight was reported in each case (Table 2).

Flatness
were cut and lengths of each strip were measured. Variation in the lengths due to non-uniformity constriction was considered to be equal to a Constriction% = (l1 -l2) / l2 × 100 (Equation 2) Where: l 1 = Initial length of each strip; l 2 l = Final length.

Scanning electron microscopy
The external morphology of the transdermal patches before and after the skin permeation experiments were analyzed by using a scanning Japan). The samples were mounted on stubs and coated with gold palladium alloy and examined under the microscope.

In-vitro drug-release study
The release rate determination is one of the most important studies that must be performed for all controlled release delivery systems. The dissolution studies of patches are very crucial, because one needs to maintain the drug concentration on the surface of stratum corneum consistently and substantially greater than the drug concentration in the body, to achieve a constant rate of drug permeation (10) dissolution of the patches was performed using six stage dissolution apparatus (I.P. /B.P/ U.S.P., Thermonik, Campbell Electronics). The patches were placed in respective baskets with their drug matrix exposed to dissolution medium, phosphate buffer (pH 7.4). All dissolution studies were performed at 50 rpm with each dissolution Samples were withdrawn at different time intervals and analyzed using a UV spectrophotometer at 322 nm against blank. Cumulative percentage of the drug-release was plotted against the time for different formulations.

In-vitro skin permeation study
The permeation studies were performed in a sectional area of 3.14 cm 2 . The recently excised abdominal skin of albino mice was taken after The adherent fatty material on the dermis side was scraped out carefully with the blunt edge of knife. The integrity of the skin sample was assured after examining with a high power microscope.
so that polymeric side facing the receiver compartment. The isolated hairless abdominal of thread in such a way that the dermal side of the skin towards the receiver compartment and stratum corneum of the skin faced the phosphate buffer (pH 7.4) and the receptor media was constantly stirred with the help of a magnetic stirrer. The temperature of the each time) were withdrawn at different time intervals and an equal amount of receptor media was replaced each time. Absorbances of the samples were read spectrophotometrically at 322 nm. The amount of drug permeated per square centimeter of skin was plotted against the time. Under similar experimental condition, another set was run without using transdermal patch as a blank.

Results and Discussion
Physicochemical characterization of the patches PVP and EC combinations have already been utilized for sustaining the release of salicylic acid (11), for sustained release microsphere of stavudine (12) and also for making transdermal patches of diclofenac diethylammonium (13). The transdermal patches of ketorolac tromethamine were subjected to various physical characterizations like moisture content, moisture and thickness. The summary of the results of physicochemical evaluations is presented in Table 2. The moisture content and moisture uptake of various formulations showed that by increasing in hydrophilic polymer (PVP), both moisture content percentage and moisture content (3.11-3.86% w/w) in the formulations helps them to maintain stable and prevents them Again, low moisture uptake protects the material from microbial contamination and bulkiness of the patch. No amount of constriction in the Table 2. Physicochemical parameters of the patches.

Weight of patch in mg ± SD (n = 25)
Run 1  ketorolac transdermal patches ensured their can maintain a smooth and uniform surface when they are administered onto skin. The thickness measurement of drug-containing patches showed the formulation of very thin patches (0.83-0.9 mm) which is an important factor to provide patient compliance. Negligible weight variation found among the transdermal patches formulated in different batches, helped in maintaining dosage uniformity.
Scanning electron microscopy SEM was done to study the surface morphology of the patches before and after the release of drug from the patches. Figure 1(a) shows the homogenous distribution of drug clusters in the matrix, before applying on skin. Figure 1(b) is the scanning electron micrograph of patch after 24 h of drug permeation. It shows the number of holes present in the patch after the release of drug clusters.

Drug-excipient interaction study
To investigate the drug-excipient interaction during formulation, the FTIR spectra of ketorolac tromethamine with or without excipients were recorded (Figure 2). In the spectrum of ketorolac tromethamine, major peaks (3,350 cm -1 [NH stretch]; 1,725 cm -1 C = O stretch (acid); 1,167 cm -1 C = O stretch (diaryl ketone); and 3,450 cm -1 [OH (acid)] were seen in subsequent spectra (Figure 2). This indicated no major interaction between the active ingredient and the excipients.

In-vitro drug release study
The sustained release performance and the reproducibility of rate and duration of drugrelease were studied in-vitro. In-vitro release advance, the way the drug will behave in-vivo (14). The rate of release of drug was found maximum (76.83 ± 2.17% drug released after 8 h) with the run-5 containing PVP alone. The rate of drug-release from other patches decreased with increase in proportion of EC and found minimum (19.27 ± 1.45%) with run-1 containing EC alone. The addition of hydrophilic component workers (15). It has also been reported that PVP decreases the crystallinity of the drug in the patch which accounts for the increased release of drug with an increase in PVP concentration in the patches (16). In addition, it can be suggested that by increasing EC concentration in the patch, drug-release can be sustained.
To investigate the mechanism of drug-release and to compare the performance of various matrix formulations, the percentage of drug-release law model (17).
Power law model is expressed as: Where M t is the amount of the drug released at time t, M is the amount of the drug released  (Table 3). In the present investigation, it was found that for runs 1 and 2, the model that

In-vitro skin permeation study
transdermal patches is presented in Figure 3. Effects of the variables on the in-vitro drug permeation from the transdermal patches were studied by statistical experimental design. Experimental design has been widely used formulation variables and their interactions with response variables (18)(19)(20). In-vitro skin permeation study is predictive of an in-vivo performance of a drug (9). The study was done through abdominal mice skins using sectional area of 3.14 cm 2 . In this study, a D-optimal mixture design ( Table 1)

Model
Run 1   ) after 24 h. Increase in the proportion of EC in the patch caused decrease in the drug release (only 19.27 ± 1.45% drug released with the transdermal patch containing EC alone; run 1), as well as provided lowest cumulative amount of drug permeation through 2 after 24 h). A correlation has been established between in-vitro skin permeation versus in-vitro release times (Figure 7).

Optimization of formulation
The optimum values of the variables were obtained by numerical analyses using the design expert software and based on the criterion of desirability (21). It optimizes any combination of one or more goals and the goals may apply to either factors or responses. The goals are combined into an overall desirability function. The program seeks to maximize this function. The goal seeking begins at a random starting point and proceeds up the steepest slope to a maximum. There may be two or more maximums because of the curvature in the response surfaces and their combination into the desirability function. By starting from several points in      the «best» local maximum. A desired goal was The optimized formulation (desirability value of 0.857; Figure 8) was achieved with 90% PVP and 10% EC. Transdermal patches with the predicted optimum levels of formulation variables were fabricated and analyzed to validate the optimization procedure. Comparative values of predicted and observed response along with the formulation components are reported in Table 4 which demonstrated that the observed values of a new batch were mostly similar with predicted values with 2.5% predicted error.

Conclusion
Transdermal system was developed and optimized for permeation parameters successfully employing the principles of Experiment Design. The physicochemical properties of the fabricated patches were found hopeful. The optimized formulation was achieved with 90% PVP and 10% EC. Further in-vivo studies in suitable animal models may be carried out with the performance. X 1 (PVP%) X 2 (EC%) Observed a Predicted Predicted error b r (%) Optimized 90.00 10.00 34.03 ( ± 1.07) 33.20 2.5 Table 4. Optimized levels for formulation variables and comparative values of predicted and observed responses for numerically optimized formulation.